Whisker Stimulation Increases the Astrocytic Participation at the Bouton–Spine Interface

<div><p>(A) Shows a 3D reconstruction of a spine head (green), its PSD (red), and the associated astrocyte (blue). The orientation of the structure shows the region occupied by the axonal bouton (removed). The line drawing below shows the parameters measured: the total perimeter of the interface between the bouton and the spine, and the part of this perimeter that is occupied by the astrocyte, the astrocytic perimeter.</p> <p>(B) Stimulation did not change the degree of contact between bouton and spine, measured by the total perimeter (<i>p</i> > 0.5). However, the amount of the perimeter occupied by the astrocyte was significantly increased (<i>p</i> < 0.0001), using mean values per animal.</p> <p>(C) Correlation between the length of the perimeter that is occupied by astrocytic membrane and the PSD surface area on spines in unstimulated (light grey diamonds, <i>n</i> = 271; <i>p</i> < 0.001, <i>R²</i> = 0.68) and stimulated neuropil (dark grey diamonds, <i>n</i> = 340; <i>p</i> < 0.001, <i>R²</i> = 0.73).</p></div

Sensory Stimulation Increases Percentage of Spines Whose Bouton–Spine Interface Is Surrounded by Astrocyte

<p>The histogram shows the distribution of four classes of spines, sorted according to their degree of contact with the astrocyte (see examples of classes I–IV), expressed as mean ± standard error of the mean (SEM) (unstimulated, <i>n</i> = 6 mice; stimulated. <i>n</i> = 6 mice). The percentage of spines in class IV, whose bouton–spine interface is completely surrounded by an astrocytic element, was increased significantly in stimulated mice (<i>p</i> < 0.03). Dendritic spines were classified into four classes, I–IV, based on the arrangement of the astrocyte at their surface. Electron micrographs of spines of each class are shown, as well as the 3D reconstruction of the whole spine to the right. (spines are indicated with an S and axonal boutons, B). Examples of spines in classes I–IV, and their reconstructions, are shown. Scale bar in lower micrograph represents 200 nm.</p

Up-Regulation of GLAST and GLT1 Protein Levels after Whisker Stimulation

<div><p>(A) A single barrel column (C2) was removed by aspiration through a glass micropipette, under sodium pentobarbital anesthesia.</p> <p>(B) Tangential section of the barrel cortex, Nissl stained, shows the location of the excised barrel column. A clear hole can be seen in the section in the region of barrel C2, with the neighboring barrels intact.</p> <p>(C) Representative immunoblot microassay of C2 columns dissected immediately after 24 h of C2 whisker stimulation (stim), 4 d after stimulation (4 d post stim), and from unstimulated mice (unstim). Blot was probed for GLAST, GLT1, and actin, and indicates an increase in GLAST and GLT1 levels after 24 h of whisker stimulation, but not 4 d later.</p> <p>(D) These changes were quantified using densitometry with the values being normalized against the actin levels. Results were expressed as percentages of levels in unstimulated mice (100%) and statistically analyzed with a Tukey studentized range test, <i>p</i> < 0.01; error bars indicate SD. Scale bar in (B) indicates 0.5 mm.</p> <p>(E) Representative immunoblots from animals treated as in (C), and analyzed for protein levels of EAAC1, tubulin, and actin.</p> <p>(F) Quantification of the immunoblot signals revealed no significant alteration in EAAC1 levels in stimulated animals. EAAC1 and tubulin values were normalized on actin levels, and expressed as % of level in control animals. Note that the relative level of tubulin was unchanged by the stimulation. Error bars indicate SD.</p></div

Serial EM Images, and Reconstruction, of a Dendritic Spine from a C2 Barrel Hollow

<div><p>(A–D) Show four micrographs from a series of 18 that were used to reconstruct the entire dendritic spine (S); making an asymmetric synaptic contact with a bouton (B) (arrow in [A] and [B]). The astrocytic element that surrounds this spine is marked with an asterisk (*) and can be seen to be closely associated with the interface between the spine head and the axonal bouton. Scale bar in (D) indicates 0.5 μm.</p> <p>(E) Shows the corresponding 3D reconstruction of this spine (green), bouton (grey), PSD (red), and astrocyte (blue) in three images below. The left-hand image shows the spine in the same orientation as the above micrographs, with a transparent astrocyte revealing the shape of the spine beneath; the middle image is in the same orientation, but the astrocyte is now opaque, showing the degree to which the spine is covered. The right-hand image shows the spine and covering astrocyte, viewed after a 180° rotation around the <i>y</i> axis.</p></div

Excitatory synaptic function is selectively enhanced in CA1 of hippocampal brain slices following <i>in vitro</i> SAHA treatment.

<p>(A) The median amplitude of mEPSCs was significantly increased in SAHA-treated slices (p<0.05, n = 14 vehicle, 14 SAHA), while there was no significant change to mEPSC frequency as measured by the median interval between events (p>0.05). Example mEPSC traces from vehicle and SAHA treated slices are shown inset (scale bar represents 10 pA and 250 ms). (B) SAHA treatment did not significantly alter the amplitude or frequency of mIPSCs (p>0.05, n = 14, 11). Example mIPSC traces from vehicle and SAHA treated slices are shown inset (scale bar represents 10 pA and 500 ms). All data points are plotted as mean ±SEM.</p

SAHA IC₅₀ values for each HDAC isozyme are shown as measured using <i>in vitro</i> enzymatic assays.

<p>Values are in µM and confidence intervals are in parentheses.</p

SAHA treated slices exhibit enhanced induction of LTP and impaired LTD.

<p>(A) An induction protocol that was subthreshold in vehicle treated slices readily evoked LTP in SAHA treated slices (p<0.05, n = 5,5). Example traces before and after LTP induction are shown in red for SAHA and black for vehicle treated slices (scale bars represent 20 pA and 20 ms). (B) An induction protocol that readily induced LTD in vehicle treated slices could not produce LTD in SAHA treated slices (p<0.05, n = 9 vehicle, 8 SAHA). Example traces before and after LTD induction are shown in red for SAHA and black for vehicle treated slices (scale bars represent 25 pA and 20 ms). Data are plotted as mean ±SEM.</p

Pharmacokinetic analysis of SAHA following i.p. injection.

<p>A) Bioanalysis of the time course of total (top) and unbound (bottom) plasma, CSF, and brain levels of SAHA following a single 50 mg/kg ip injection (n = 3 mice/time point). The dotted red lines represent the SAHA concentration imposed on the <i>in vitro</i> slice cultures for the electrophysiological studies. B) Total (top) and unbound (bottom) SAHA levels are shown following a 150 mg/kg ip injection (n = 3/time point). All data is shown as mean ± SD.</p

Fear memory deficits in Tg2576 mice are not rescued by SAHA treatment.

<p>A) Compared to non-transgenic littermates (n = 15), Tg2576 (n = 14) mice showed significantly less freezing than wt mice when returned to the context in which conditioning occurred (context, p<0.001), when placed in an altered context (altered, p<0.01), or in response to the cue used for conditioning (cue, p<0.05). B) Tg2576 mice were treated daily for 33 days prior to, as well as during fear conditioning with either vehicle (n = 14), 25 mg/kg SAHA (n = 13), or 50 mg/kg SAHA (n = 13). There was no effect of treatment on the percentage of time Tg2576 mice spent freezing in response to the context, altered context, or cue (p>0.05). C) There was no effect of treatment on the percentage of time spent freezing during conditioning (p>0.05). D) There was no effect of treatment on the distance traveled in the open field test (total distance = 38.0±7.7 m for vehicle, 39.6±6.1 m for 25 mg/kg SAHA, and 34.2±5.2 m for 50 mg/kg SAHA, p>0.05). All data are plotted as mean ±SEM.</p

Acute or chronic SAHA treatment does not produce significant drug class activity signatures as assessed by the SmartCube®.

<p>A. Groups of mice were treated acutely with a single injection of 50 mg/kg or 150 mg/kg SAHA or vehicle. In addition, a group was treated with valproate (225 mg/kg). Both does of SAHA were behaviorally inactive without a clear therapeutic signal. In contrast valproate was behaviorally active (p<0.001, discrimination index = 100%) with a strong anxiolytic signature and a mild psychostimulant signature. B. Groups of mice were treated daily for 14 days with SAHA or Valproate. While the lower dose of SAHA appeared behaviorally active (p<0.001, discrimination index = 88%), the activity was not consistent with any known therapeutic signal and the higher dose was not behaviorally active. In contrast valproate showed a strong behavioral activity (p<0.001, discrimination index = 98%) with a predominantly anxiolytic signature. C. The legend shows the 15 classes of behavioral activity that were assessed.</p